Antibody conjugates with macrocyclic ligands

ABSTRACT

The conjugation of antibodies to a macrocyclic conjugate compound wherein the conjugate compound has the structure (I), wherein R 1  is --(CH 2 ) p  --R 6  --CH 2 ) q  -- where p and q are the same or different and are 0, 1 or 2, and --R 6  -- is --((CH 2 ) n  --, where n is 0 or 1, --NH--, --O--, --S-- or (II), R 1  optionally being alkyl substituted, provided that neither p nor q is 0 unless R 6  is --CH 2  --; R 2  are --CH 2  CH 2  -- or --CH 2  CH 2  CH 2  --, optionally alkyl, alkoxyalkyl or hydroxyalkyl substituted; R 3  are the same or different and are --H, alkyl, hydroxyalkyl, alkoxyalkyl, carboxyalkyl, carboxyalkyl ester, phosphate, sulphonate or phosphonate; R 4  is one of the compounds of formula (III) optionally alkyl substituted, wherein R 7  is --H, alkyl, hydroxyalkyl, or alkoxyalkyl provided that when R 4  is (III d), R 3  is not carboxyalkyl, R 5  is a linker, and Ab is an antibody. The conjugate compound provides for complexes of metals such as Tc, Co, Re, Cu, Au and Ag which are useful reagents for in vivo imaging and therapy.

The present application is a file wrapper continuation of U.S.application Ser. No. 07/772,777 filed 10/7/91 now abandoned; which is adivisional of U.S. application Ser. No. 07/132,955 filed 12/14/87 whichissued to U.S. Pat. No. 5,087,696 on 2/11/92.

FIELD OF THE INVENTION

This invention relates to a conjugate compound comprising a macrocyclicligand and an antibody, to a metal complex of the conjugate compound andto formulations of the metal complex for use in therapy and diagnosis.

BACKGROUND TO THE INVENTION

It is known to label an antibody with a metal atom, in order to targetthe metal atom to a specific tissue type, both in vitro and in vivo.Such labelled antibodies have applications in locating specific tissuetypes (e.g. employing computer-aided tomographic techniques where themetal atom is in some way detectable) and in the treatment of celldisorders (e.g. treating mammalian tumours where the metal atom is acytotoxic radionucleide).

Conventionally, the metal atom has been complexed to a conjugatecompound comprising a ligand covalently attached to an antibody. Theligand may be, for example, an acyclic chelate such as a substituteddiethylenetriaminepentaacetic acid (DTPA) (Gansow O. A. et al, Inorg.Chem., (1986), 25, 2772) or ethylenediaminetetraacetic acid (EDTA)(Meares, C. F. et al, Acc. Chem. Res., (1984), 17, 202). Such acyliccomplexes however tend to be unstable in vivo either as a result ofacid-catalysed decomplexation or competitive chelate binding by Ca²⁺ orZn²⁺ in serum or as a result of competition from transferrin (Moerlein,S. M. et al, Int. J. Nuc. Med. Biol., (1981), 8, 277). The lack ofstability can result in uncomplexed metal atoms in the body which have acytotoxic effect on healthy tissue or which markedly reduce thesignal-to-noise ratio of an imaging technique. The use of macrocyclicligands in the labelling of antibodies has been suggested in broad terms(Gansow, O. A. et al, Am. Chem. Soc. Symp. Ser., (1984), 241, 215;Published British patent application GB2122641A; Meares, C. F. et al,Anal. Biochem., (1985), 148, 249-253)).

The object of the present invention is to provide improved conjugatecompounds involving macrocyclic ligands capable of binding metals togive complexes which are stable, in vivo.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided aconjugate compound having the following structure: ##STR1## wherein R¹is --(CH₂)_(p) --R⁶ --(CH₂)_(q) -- where p and q are the same ordifferent and are 0, 1 or 2, and --R⁶ -- is --(CH₂)_(n) --, where n is 0or 1, --NH--, --O--, --S-- or ##STR2## R¹ optionally being alkylsubstituted, provided that neither p nor q is O unless R⁶ is --CH₂ --,

R² are --CH₂ CH₂ -- or --CH₂ CH₂ CH₂ --, optionally alkyl, alkoxyalkylor hydroxyalkyl substituted,

R3 are the same or different and are --H, alkyl, hydroxyalkyl,alkoxyalkyl, carboxyalkyl, carboxyalkyl ester, phosphate, sulphonate orphosphonate

R⁴ is ##STR3## optionally alkyl substituted, wherein R₇ is --H, alkyl,hydroxyalkyl, or alkoxyalkyl provided that when R⁴ is --CH₂ --CH--CH₂--, R³ is not carboxyalkyl,

R⁵ is a linker, and

Ab is an antibody.

The conjugate compound of the invention provides for complexes of metalsor such as Tc, Co, Re, Cu, Au and Ag Pb, Bi, In and Ga which are usefulreagents for in vivo imaging and therapy. Particularly preferred arecomplexes of Tc and Cu.

R¹ is preferably --CH₂ CH₂ CH₂ -- or --CH₂ CH₂ --O--CH₂ CH₂ --. Theradicals R² are preferably the same and may be --CH₂ CH₂ --. In eachcase, one or more hydrogen atoms may be replaced with an alkylhydroxyalkyl or alkoxyalkyl radical, such as lower alkyl radical, forexample methyl.

The aromatic ring, where present, in R⁴ may be 1,3,5 or 1,3,4substituted depending to the ring size required and the synthetic routeadopted to make the compound.

R⁴ may be substituted with lower alkyl such as methyl. R⁷ is preferably--H or lower alkyl (C₁ -C₃) such as methyl, but may be higher alkyl suchas C₄ -C₁₅ alkyl.

The linker, R⁵, may be any diradical adapted to link the antibody to themacrocyclic part of the conjugate compound such that the bindingaffinity and specificity of the antibody are not substantially impairedand such that the macrocyclic part of the conjugate molecule maycoordinate with a metal, such as a transition metal or a B-metal.

The antibody, Ab, may be a complete antibody molecule or a fragmentthereof or an analogue of either of these, provided the antibody Abcomprises a specific binding region, for example a Fab' or F(ab)₂ 'fragment. The antibody Ab may be a polyclonal or a monoclonal antibodyor a fragment thereof. Thus the antibody Ab may be obtained from ahybridoma cell line or other animal cell line. Preferably the antibodyAb has specificity for a mammalian tumour.

In a second aspect of the invention there is provided a conjugatecompound of the invention including a complexed metal atom. The metal ispreferably a metal selected from Tc, Re, Co, Cu, Au, Ag, Pg, Bi, In andGa. Particularly preferred are complexes of Tc and Cu. The metal issuitably a radioactive isotope.

The radioisotopes of technetium (Tc) and rhenium (Re) provide usefulreagents for imaging and therapy. In particular, ^(99m) Tc is a strongγ-emitter and, as such is useful in γ-ray imaging. ^(186/188) Re is agood β-emitter and is useful for β-particle therapy. Both are readilyavailable from a generator as the MO₄ anion, and may be complexed in areduced form to provide complexes which are stable in body fluids.

In a preferred form of the first aspect of the invention there isprovided a conjugate compound having the structure: ##STR4## wherein R³,R⁵ and Ab are as defined for structure I above R³ is preferably --H.

In a preferred form of the second aspect of the invention there isprovided a conjugate compound of structure II complexed with atechnetium, copper or rhenium atom, preferably ^(99m) Tc, ⁶⁷ Cu, ⁶⁴ Cu,or ^(186/188) Re.

In a further preferred form of the first aspect of the invention thereis provided a conjugate compound having the structure: ##STR5## whereinm is 1 to 5 and R³, R⁵ and R⁷ are as defined for structure I above.

The conjugate compound of structure III is suitable for complexing arange of metal atoms, including Tc, Cu, Co, In, Au and Ag, optimumbinding being achieved by selection of the ring size by adjustment ofparameter m and by adjusting the potential coordination number by choiceof radical R³.

In a further preferred second aspect of the invention, there is provideda conjugate compound of structure III wherein m is 3 and R³ is --H or--CH₃ complexed with a technetium, rhenium, copper or gold atom,preferably ^(99m) Tc, ⁶⁷ Cu, ⁶⁴ Cu, ¹⁹⁹ Au, ^(186/188) Re.

The conjugate compound of the second aspect of the invention may be usedas a pharmaceutical, for example, in the treatment of mammalian tumoursor as a diagnostic reagent for use in imaging in vivo as a tracer.

In a third aspect, the invention provides a composition comprising aconjugate compound of the second aspect of the invention or an additionsalt thereof and a pharmaceutically acceptable carrier. The compositionmay be used in the treatment of tumours or as a diagnostic reagent forimaging in vivo.

In a fourth aspect, the invention provides a compound of the followinggeneral structure: ##STR6## wherein R¹, R², R³ and R⁴ are as defined forstructure I above and R⁸ comprises a reactive group covalently attachedto R⁴. The reactive group provides a point of attachment for an antibody(Ab) either directly, or through a linker. R⁸ may comprise --(CH₂)_(n)--NH₂, --(CH₂)_(n) --Ar--(CH₂)_(m) --NH₂, --(CH₂)_(n) --CO₂ H,--(CH₂)_(n) --Ar--NCS, or --(CH₂)_(n) NHCOR⁹, wherein n and m=0 to 4 andare the same or different and R⁹ is a haloalkyl such as --CH₂ Br. Theisothiocyanate and haloalkyl functionalised cycles may be linkeddirectly to an antibody thiol or amino group. Most preferably R⁸ is aprimary amine such as --CH₂ --Ar--CH₂ NH₂.

It has been discovered that an exocyclic primary amine having a pKa from8 to 10 exhibits exceptional qualities of selectivity allowing theattachment of linkers to the macrocycles of the invention withoutsubstantial disadvantageous side reactions and polymerisation resultingfrom the existence of the saturated ring nitrogens. The selectivityeffect is optimised by careful pH control. Suitably, the attachment of alinker to compound IV is conducted at from pH 6 to pH 8. The primaryamine may be conveniently reacted with an acylating agent, such as anactivated ester moiety on a linker compound.

The linker comprises a moiety for reaction with R⁸ and a moiety forattachment to antibody. Suitably the moiety for attachment to antibodyis a thiol-specific group such as a maleimide group or a vinyl groupconjugated with an aromatic nitrogen heterocycle (e.g. a vinyl pyridinegroup).

In a preferred aspect of the invention, a primary amine group iscovalently linked to a maleimide or vinyl pyridine-containing linkercompound, which may be subsequently reacted with a thiol group on theantibody or modified antibody. This compound therefore provides animportant intermediate in the production of a conjugate compoundaccording to the first or second aspect of the invention.

The invention further provides addition salts of the conjugate compoundsdescribed.

The invention further provides a method for preparing conjugatecompounds of the first aspect of the invention comprising reactingtogether a compound of the fourth aspect of the invention with a linkerand an antibody or an antibody fragment.

The invention further provides a method for the treatment of a diseasedcell comprising administering an effective amount of a conjugatecompound according to the second aspect of the invention, the conjugatecompound comprising an antibody having specificity to the diseased cellsand the macrocycle including a metal cytotoxic to the diseased cells.The diseased cells may be, for example, mammalian tumour cells.

The invention further provides a method of tumour imaging comprisingadministering an effective amount of a conjugate compound of the secondaspect of the invention, the conjugate compound comprising an antibodyto the tissue and the macrocycle including a detectable metal. Thetissue may be for example, mammalian tumour cells.

The invention is illustrated by the following Examples.

Description of Specific Embodiments EXAMPLE 1

A synthesis was performed of a macrocyclic compound having thestructure: ##STR7##

To a solution of sodium ethoxide (0.051M) in dry ethanol (100 cm³) undernitrogen, was added diethyl malonate (16 g, 100 mmol) in dry ethanol (35cm³) and the mixture was stirred at room temperature (0.5 h). To thecooled solution was added a solution of α-bromo-paratolunitrile (10 g,0.051 mol) in dimethylformamide (50 cm³) and the mixture was refluxed(24 h). After cooling to room temperature, distilled water (150 cm³) wasadded, the mixture filtered and the filtrate was extracted with di ethylether (3×50 cm³). The ether was removed under reduced pressure and theresidue distilled to yield p-cyanobenzyldiethylmalonate (6 g, 45%), bp150° . (0.01 mm) (δ_(H) CDCl₃) 7.59(2H, d, Hr); 7.33 (2H d, Ar); 4.16(4H, mult, CH₂ O) 3.63 (1H,t, CHCH₂), 3.27 (2H, d, CHCH₂); 1.22 (6H, t,CH₃). (δ_(c) CDCl₃) 167.3(COEt); 143.3, 132.1, 129.5, 116.5(Ar); 110.5(CN); 61.6 (CH₂ O); 53.0 (CO₂ Et)₂); 34.4 (CH₂ --Ar); 13.8(CH₃).

A mixture of 1,3,8,11-tetraazaundecane (2.77 g, 17.3 mmol) andp-cyanobenzyldiethylmalonate (4.75 g, 17.3 mmol) in dry ethanol (50 cm³)was refluxed for five days, evaporated to dryness and the residuechromatographed on silica gel (0.063 0.20 mm, Merck) gradient elutingwith an aqueous ammonia/methanol dichloromethane solvent of compositionwhich was initially 1:12:87 and finally 6:44:50. The required diamidewas isolated as a colourless solid (1.1 g, 19%), mp 209°-211° C. (δ_(H)CDCl₃) 7.56 (2H,d,Ar), 7.33 (2H,d,ArH), 6.65 (2H, brs, NHCO), 3.50 (2H,mult, CH₂ Ar), 3.25 (4H, mult., CH₂ NCO), 1.78 (2H, brs, NH), 1.65 (2Y,mult., CH₂ C) m/e (isobutane DCI) 344 (m⁺ =1) IR (KBr) ν 3290 (NH),2910, 2805, (CH), 2225 (C=N), 1638(NCO), 1530 (NH) Rf(SiO₂ : aq.NH₃/MeOH/CH₂ Cl₂) (6:44:50) 0.25.

To the diamide (0.97 g, 2.8 mmol) in tetrahydrofuran (25 cm³) was addedby syringe under nitrogen a borane-tetrahydrofuran solution (47 cm³,1.0M) and the mixture refluxed (24 h). After destroying the excessborane by careful addition of methanol (4 cm³), the solution wasevaporated to dryness and the residue treated with hydrochloric acid(6M, 30 cm³) and refluxed for 3h. On cooling, the solution was basifiedwith sodium hydroxide (2.5 cm³) and extracted with chloroform (3×50cm³). Removal of solvents under reduced pressure gave a colourlessresidue which was recrystalised from toluene to give the desiredpentamine (Va) as a colourless solid (0.75 g, 83%), mp 149°-150° C.(δ_(H) CDCl₃) 7.21 (2H,d,ArH), 7.14(2H,d,ArH), 3.83(2H,d,CH₂ Ar) 2.82.4(19H, mult., CH₂ Ar+ CH₂ N), 2.2 (6H,brs,NH), 1.71 (2H,quint, CH₂ C)m/e (isobutane DCI) 320 (m⁺ =1), 319 (M⁺) (δ_(c) CDCl₃) 140.9 (ArCH₂ -Caryl) 139.0 (CH₂ -C(aryl); 129.1, 127.0 (rCH); 55.8, 50.72, 49.3 46.1(CH₂ N); 40.8 (C, 38.6 CH₂ -Ar); 29.3 (CH₂).

To solution of the p-nitrophenyl ester of 2-vinyl, 6- methoxy aceticacid pyridine (31.2 mg. 0.1 mmol) in p-dioxan (5 cm³) was added asolution of Va (31.9 mg, 0.1 mmol) in buffered water (pH 6.8, 5 cm) 0.5Min 1,4-piperazine bis (ethanesulphinic acid) [PIPES]) and the; solutionstirred at 20° C. for 3 r. Purification of the ester by ion-exchangeHPLC gave the desired ester Vb (44.5 mg, 90%). m/e (fast atombombardment, glycerol/H₂ O) 495 (M⁺ 1), 494.33 (m⁺), 304, 224, 185, 115.(δ_(H) D₂ O as the diacetate salt) 7.76 (1H,t,pyCH,J 8.Hz₂); 7.48(1H,d,pyCH); 7.30 (1H,d,pyCH); 7.17 (3H,brs,ArCH); 6.74 (1H,d,vinylCH);6.03(1H,d,J 17.7); 5.50 (1H,d,J 11.0); 4.64 (2 Y, COCH₂ O); 4.33 (2H₃ S₃CH₂ O); .15 (2H,S, CH₂ NH); 2.98-2.32 (19Y, mult. CH₂ N+CH₂ Ar+CHCH₂),1.84 (2Y, mult, CH₂ CH₂ CH₂).

The maleimide fuctionalised macrocyle Vc may be made in a similar mannerto that described for Vb, using the p-nitrophenyl orN-hydroxysuccinimide ester of N-(4-carboxycyclohexylemthl)-malemide,(Yamada, et al, Eur. J. Biochem, (1979), 101, 395).

In order to link the functionalised macrocycle to the antibody, theantibody was first reacted with Traut's reagent (2-iminotheiolane) togive free thiol groups for attachment. For example, a solution of Lym-1(2 mg) in 0.2M sodium phosphate buffer (pH 7.4) was mixed with 75 μdm³of 2-iminothiolane (20 mM) in 50 mM triethanolamine-HCl (pH 8) andB-thioethanol (6 μdm³) was added. After incubating at 4° C. (1h), themodified antibody was purified on a G-10 Sepharose column. To a solutionof the antibody at pH 6.8 (PIPES buffer) was added the vinyl-pyridineconjugated macrocycle, (Vb), and the reaction was allowed to proceed for3 hours at room temperature. The resulting macrocycle-linker-antibodyconjugate was purified on a G-10 Sepharose column.

Complexation of Vb or Vc with technetium was effected usingmodifications of the reported literature methods (Troutner el al Int. J.Appl. Radiat Isot. (1982), 33,891; Childs R. L. et al, U. Nucl. Med.,(1985), 26, 193) using TcO₄ ⁻ in the presence of stannous tartrate andusing a phosphate or succinate buffer, in order to facilitate transferof ^(99m) Tc from reduced technetium (V) to the macrocycle.

Complexation of rhenium was achieved by reaction of equimolar quantitiesof Vb and ReOCl₃ (PPH₃)₂ (Johnson et al, J. Chem. Soc., (1964), 1054) inchloroform.; After 5 min. at 25° C., diethylether was added and aReOCl(Vb)²⁺ complex precipitated. In aqueous solution this existsprimarily as the ReO₂ (Vb)⁺ complex, as the chlorine trans to the Re-oxobond is labile.

A solution of ⁶⁴ CuCl₂ (30 μl), was added to the macrocycle conjugate[200 μl with approx. 3 mg Ab/ml] in 0.1M sodium acetate or 0.1M sodiumsuccinate at a pH of 6.0. The solution was incubated at 35° C. for 0.5y, and then chromatographed on a Sephadex G-50 (fine) gel column [whichhad been previously swollen (overnight) in the PBS buffer used forprotein eluting] of dimension 1 cm×6 cm, collecting 0.5 ml aliquots andcounting each fraction in order to separate the desired copper-boundantibody macrocycle complex.

EXAMPLE 2

A synthesis was performed of a macrocyclic compound having thestructure: ##STR8##

6- Cyanocoumarin (3.46 g, 20 mmol), prepared from 6-aminocoumarin viadiazotisation followed by reaction with cuprous cyanide (Morgan E. T.,et al, J. Chem. Soc. (1904), 1230), was added to a solution of1,9-diamino-3, 7-diazononane (3.20 g, 20 mmol) in methanol (200 cm³) andthe mixture was refluxed under argon for 6 days following the method ofKimura (J. Chem. Soc. Commun. (1985), 335). After evaporation of themethanol, the residue was chromatographed on silica as described inExample 1, and the resultant amide was reduced using excessborane-tetrahydrofuran to give VI which was purified byrecrystallisation from hot toluene, or using reverse phase HPLC methods.

The macrocycle was linked to a maleimide or a vinyl-pyridine linker asdescribed in Example 1, and the functionalised macrocycle linked to anantibody Fab' or F(ab)₂ ' fragment as described above. Complexation oftechnetium or rhenium s(V) was achieved under reducing conditions fromthe corresponding salt, using the methods disclosed in Example 1.

EXAMPLE 2

A synthesis was performed of a macrocyclic compound having thestructure: ##STR9##

Sodium metal (115 mg, 5 mmol) was dissolved in dry methanol (100 cm³)and N, N¹ -3,7-tetra(p-toluenesulphonyl)3,7-diazanonane-1,9 diamine(1.95 g, 2.51 mmol) was added as a solid. The mixture was refluxed undernitrogen for 1 hours, and solvent was removed to yield a solid disodiumsalt. After removing traces of methanol in vacuo, the solid wasdissolved in dry dimethylformamide (50 cm³) and the solution was heatedto 80° C. A solution of 6,6-dibromemethyl-3-nitrobenzine (747 mg, 2.5mmol) in dimethylformamide (20 cm³) was added over a period of 1 hour.After stirring for 2 hours at 80° C. water (40 cm³) was added over aperiod of 15 minutes and the mixture was stirred for 16 hours at 20° C.The resulting oily product was separated, dissolved in dichloromethane(50 cm³), washed with water (3× 15 cm³) and the solvent was evaporated.The cyclic tetratosylate was purified by precipitation from CH₂ Cl₂hexane (1:4 v/v) and subsequently by chromatography on a neutral columneluting with dichloromethane/methanol, (1%), to yield a tosylatedmacrocycle as a colourless glass. Detosylation was effected by addingphenol (2 g) and hydrogen bromide in acetic acid (45%, 100 cm³) to themacrocycle and stirring for 24 hours at 80° C. The resulting mixture wascooled and the solvent was removed under reduced pressure, addingtoluene to assist the removal of the acetic acid. The residue wasdissolved in water (100 cm³) and washed with dichloromethane (4×50 cm³).The aqueous phase was evaporated and the product was redissolved inwater (100 cm³) and passed down an anion exchange resin (OH⁻ form,Amberlite IR400). the aqueous solution thus obtained was evaporated,leaving a product which was crystallised as the hydrochloride salt formethanol/hydrochloric acid. The amine was regenerated by passage throughan anion exchange column, and is a pale yellow oil. Reduction of thearomatic nitro group to the corresponding amino group was effected bydissolving the free amine (618 mg, 2 mmol) in ethanol, adding palladiumon carbon (100 mg, 10%) and hydrogenating under 2 atmospheres of 30° C.for 3 hours. After filtering to remove spent catalyst, the solvent wasremoved to yield compound VI which was stored at -30° C. under nitrogen.

Complexation of Re was achieved as described in Example 1.

EXAMPLE 4

A synthesis was performed of a macrocyclic compound having thestructure: ##STR10##

The synthesis was essentially as for macrocyclic compound VII describedin Example 3, except that the disodium salt of N,N¹-3,9-tetra(p-toluenesulphonyl)-6-oxa- 3,9-diazadecane-1,11-diamine wasused (Dietrich, M. W. et al, Helv. Chim. Acta, (1983), 66, 1262) andacetate side chains were added to the ring nitrogen atoms of the freeamine prior to reduction of the aromatic nitro group.

Sodium hydroxide (1.4 g, 37.5 mmol) in water (10 cm³) was added tobromoacetic acid (2.42 g, 17.5 mmol) in water (10 cm³) at below 5° C.The free amine in ethanol (10 cm³) was added and the temperature washeld at 70° C. for 2 hours. After cooling, the solution was brought toabout pH 3.5 with hydrochloric acid, when a white precipitate of thesubstituted macrocycle VIII was formed. This was filtered,recrystallised from water and dried in a vacuum.

Complexation with Pb²⁺ was effected under aqueous conditions by mixingequimolar concentrations of lead nitrate and the macrocyle.

EXAMPLE 5

A synthesis was performed of a macrocyclic compound having thestructure: ##STR11##

EXAMPLE 5

A synthesis was performed of a macrocyclic compound having thestructure: ##STR12##

6-nitrocoumarin (9.55 g, 50 mmol) was added to a solution1,9-diamino-3,7-diazanonane (8 g, 50 mmol) in methanol (200 cm³) underargon, and the mixture was refluxed for 6 days. After evaporation of themethanol, the residue was chromatographed on silica, eluted with CH₂ Cl₂/CH₃ OH/NH₄ OH (70:27:3 v/v) to yield a yellow oil which was furtherpurified by recrystallisation from ethanol/hydrochloric acid to give atrihydrochloride salt of the cyclic amide. Successive reduction of theamide with borane-dimethylsulphide, O-methylation of the phenolic --OHgroup (under nitrogen) and reduction of the aromatic nitro group usingpalladium on carbon as described in Example 3 above resulted inmacrocycle IX.

Complexation of technetium was effected using techniques known in theliterature employing (NH₄)TcO₄ and the macrocycle in the presence of Na₂S₂ O₄ or stannous tartrate.

EXAMPLE 6

A synthesis was performed of a macrocyclic compound having thestructure: ##STR13##

The synthesis was essentially as for macrocyclic compound IX except thatacetate side claims were added to the ring nitrogen atoms, as describedin Example 4, prior to reduction of the aromatic nitro group.

Complexation of indium and gallium is effected by reaction of themacrocycle in water with InCl₃ or GaCl₃ solution in 0.04M HCl undercontrolled pH using acetate or citrate buffer for 45 minutes (see forexample Buckley, R. G. et al, Febs. Lett., (1984), 166, 202 and Meares,C. F. et al, J. Protein, Chem., (1984), 3, 215).

The macrocycle produced in Example 1, 2, 3 or 4 may be covalently boundto an antibody or an antibody fragment, using standard techniques,before or after complexation with the metal. For example a Fab'-linkermacrocycle conjugate according to the present invention may be preparedsubstantially as described below for the model macrocyle,14-(5-aminophenyl)-1, 4, 8, 11-tetraazocyclotetradecane.14-(5-aminophenyl)-1, 4, 8, 11-tetraazocyclotetra decane (1m mole) wasdissolved in 0.2M phosphate buffer (pH 7.4) and treated with theheterobifunctional cross-linking reagentsuccinimidyl-4-(p-maleimidophenyl) butyrate (1m mole) dissolved in 0.2Mphosphate buffer (pH 7.4), for two hours at room temperature. Theresulting macrocycle-linker was purified using silica gel columnchromatography, substantially as decribed by Kimura et al, (Jnl. Chem.Cos. Chem. Comm., (1985), p 385).

A monoclonal antibody was treated with the enzyme pepsin to generate aF(ab')₂ fragment. The f(ab')₂ fragment was then purified using gelpermeation chromatography. The purified F(ab')₂ fragment was thentreated with the reducing agent dithiothreitol (1m M) at pH 8 for threehours at room temperature. The resulting Fab' fragments were thenacidifed and passed through a G.10 Sepharose column to yield pure Fab'fragments. The resulting Fab' (0.1m mole) was then dissolved in 0.2Mphosphate buffer (pH 7.4) and added to a solution of themacrocycle-linker conjugate (1m mole) in 0.2M phosphate buffer (pH 7.4).

The Fab' fragment and macrocycle-linker conjugate were then allowed toreact for three hours under argon at room temperature and the resultingmacrocycle-liner-Fab' conjugate purified on a G.10 Sepharose column.

It will be understood that the invention is described by way of Exampleonly and modifications of detail may be made within the scope of theinvention.

What is claimed is:
 1. A conjugate of a monoclonal or polyclonalantibody or antigen binding fragment thereof covalently bound, eitherdirectly or through a divalent linking group, to a macrocyclic ligand ofthe formula: ##STR14## wherein n has a value of from 0 to 4;R¹ is --CH₂--CH₂ --CH₂ -- or --CH₂ --CH₂ --O--CH₂ --CH₂ --; and R⁴ is ##STR15## Abis a monoclonal or polyclonal antibody or antigen binding fragmentthereof.
 2. A conjugate according to claim 1 wherein said macrocyclicligand has the formula: ##STR16##
 3. A conjugate according to claim 1wherein said macrocyclic ligand has the formula: ##STR17##
 4. A complexof a conjugate according to claim 1 with a metal atom.
 5. A complexaccording to claim 4 wherein the metal atom is selected from the groupconsisting of technectium, rhenium, cobalt, copper, gold, and silver. 6.A composition comprising a member selected from the group consisting ofa conjugate according to claim 1 and a complex thereof with a metal atomin combination with a pharmaceutically acceptable carrier.
 7. A couplingagent of the formula: ##STR18## wherein n has a value of from 0 to 4;R¹is --CH₂ --CH₂ --CH₂ -- or --CH₂ --CH₂ --O--CH₂ --CH₂ --; and R⁴ is##STR19##
 8. A coupling agent according to claim 7 having the formula:##STR20##
 9. A coupling agent according to claim 7 having the formula:##STR21##